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991.
Unfolding usually leads to the loss of the biological function of a protein. Here, we show that an unfolding reaction activates the gene-3-protein of the filamentous phage fd for its function during the infection of Escherichia coli. Before infection, the gene-3-protein is in a fully folded locked form, in which the binding site for the phage receptor TolA is buried at the domain interface. To expose this binding site, the gene-3-protein must be activated, and previously we identified the cis-to-trans isomerization at Pro213 in the hinge region between the two domains as a key step of activation. We now report that Pro213 isomerization destabilizes the protein and leads to a loss of folded structure, presumably in the hinge region. The partially unfolded form of the gene-3-protein is metastable, and trans-Pro213 arrests the protein in this activated form for an extended time, long enough to find the receptor TolA. The partial unfolding and its timing by prolyl isomerization are essential for the biological function. 相似文献
992.
The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid 总被引:1,自引:0,他引:1
Findeisen F Linder JU Schultz A Schultz JE Brügger B Wieland F Sinning I Tews I 《Journal of molecular biology》2007,369(5):1282-1295
The universal secondary messenger cAMP is produced by adenylyl cyclases (ACs). Most bacterial and all eukaryotic ACs belong to class III of six divergent classes. A class III characteristic is formation of the catalytic pocket at a dimer interface and the presence of additional regulatory domains. Mycobacterium tuberculosis possesses 15 class III ACs, including Rv1264, which is activated at acidic pH due to pH-dependent structural transitions of the Rv1264 dimer. It has been shown by X-ray crystallography that the N-terminal regulatory and C-terminal catalytic domains of Rv1264 interact in completely different ways in the active and inhibited states. Here, we report an in-depth structural and functional analysis of the regulatory domain of Rv1264. The 1.6 A resolution crystal structure shows the protein in a tight, disk-shaped dimer, formed around a helical bundle, and involving a protein chain crossover. To understand pH regulation, we determined structures at acidic and basic pH values and employed structure-based mutagenesis in the holoenzyme to elucidate regulation using an AC activity assay. It has been shown that regulatory and catalytic domains must be linked in a single protein chain. The new studies demonstrate that the length of the linker segment is decisive for regulation. Several amino acids on the surface of the regulatory domain, when exchanged, altered the pH-dependence of AC activity. However, these residues are not conserved amongst a number of related ACs. The closely related mycobacterial Rv2212, but not Rv1264, is strongly activated by the addition of fatty acids. The structure resolved the presence of a deeply embedded fatty acid, characterised as oleic acid by mass spectrometry, which may serve as a hinge. From these data, we conclude that the regulatory domain is a structural scaffold used for distinct regulatory purposes. 相似文献
993.
The thiamine-dependent E1o component (EC 1.2.4.2) of the 2-oxoglutarate dehydrogenase complex catalyses a rate-limiting step of the tricarboxylic acid cycle (TCA) of aerobically respiring organisms. We describe the crystal structure of Escherichia coli E1o in its apo and holo forms at 2.6 A and 3.5 A resolution, respectively. The structures reveal the characteristic fold that binds thiamine diphosphate and resemble closely the alpha(2)beta(2) hetero-tetrameric E1 components of other 2-oxo acid dehydrogenase complexes, except that in E1o, the alpha and beta subunits are fused as a single polypeptide. The extended segment that links the alpha-like and beta-like domains forms a pocket occupied by AMP, which is recognised specifically. Also distinctive to E1o are N-terminal extensions to the core fold, and which may mediate interactions with other components of the 2-oxoglutarate dehydrogenase multienzyme complex. The active site pocket contains a group of three histidine residues and one serine that appear to confer substrate specificity and the capacity to accommodate the TCA metabolite oxaloacetate. Oxaloacetate inhibits E1o activity at physiological concentrations, and we suggest that the inhibition may allow coordinated activity within the TCA cycle. We discuss the implications for metabolic control in facultative anaerobes, and for energy homeostasis of the mammalian brain. 相似文献
994.
Santos-Silva T Dias JM Dolla A Durand MC Gonçalves LL Lampreia J Moura I Romão MJ 《Journal of molecular biology》2007,370(4):659-673
Sulphate-reducing bacteria have a wide variety of periplasmic cytochromes involved in electron transfer from the periplasm to the cytoplasm. HmcA is a high molecular mass cytochrome of 550 amino acid residues that harbours 16 c-type heme groups. We report the crystal structure of HmcA isolated from the periplasm of Desulfovibrio gigas. Crystals were grown using polyethylene glycol 8K and zinc acetate, and diffracted beyond 2.1 A resolution. A multiple-wavelength anomalous dispersion experiment at the iron absorption edge enabled us to obtain good-quality phases for structure solution and model building. DgHmcA has a V-shape architecture, already observed in HmcA isolated from Desulfovibrio vulgaris Hildenborough. The presence of an oligosaccharide molecule covalently bound to an Asn residue was observed in the electron density maps of DgHmcA and confirmed by mass spectrometry. Three modified monosaccharides appear at the highly hydrophobic vertex, possibly acting as an anchor of the protein to the cytoplasmic membrane. 相似文献
995.
996.
Van Roey P Pereira B Li Z Hiraga K Belfort M Derbyshire V 《Journal of molecular biology》2007,367(1):162-173
The 440 amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168 residue mini-intein, DeltaI-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation (DeltaI-CM) increased C-terminal cleavage activity. Using the DeltaI-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139 residue mini-intein, DeltaDeltaI(hh)-SM, by replacing 36 amino acid residues in the residual endonuclease loop with a seven-residue beta-turn from the autoprocessing domain of Hedgehog protein. The three-dimensional structures of DeltaI-SM, DeltaDeltaI(hh)-SM, and two variants, DeltaDeltaI(hh)-CM and DeltaDeltaI(hh), have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N and C termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side-chain of Asp422 plays a role in both N and C-terminal cleavage, thereby suggesting that this highly conserved residue regulates the balance between the two reactions. 相似文献
997.
The plasmid R1162 encodes proteins that enable its conjugative mobilization between bacterial cells. It can transfer between many different species and is one of the most promiscuous of the mobilizable plasmids. The plasmid-encoded protein MobA, which has both nicking and priming activities on single-stranded DNA, is essential for mobilization. The nicking, or relaxase, activity has been localized to the 186 residue N-terminal domain, called minMobA. We present here the 2.1 A X-ray structure of minMobA. The fold is similar to that seen for two other relaxases, TraI and TrwC. The similarity in fold, and action, suggests these enzymes are evolutionary homologs, despite the lack of any significant amino acid similarity. MinMobA has a well- defined target DNA called oriT. The active site metal is observed near Tyr25, which is known to form a phosphotyrosine adduct with the substrate. A model of the oriT substrate complexed with minMobA has been made, based on observed substrate binding to TrwC and TraI. The model is consistent with observations of substrate base specificity, and provides a rationalization for elements of the likely enzyme mechanism. 相似文献
998.
Boutz DR Cascio D Whitelegge J Perry LJ Yeates TO 《Journal of molecular biology》2007,368(5):1332-1344
A growing number of organisms have been discovered inhabiting extreme environments, including temperatures in excess of 100 degrees C. How cellular proteins from such organisms retain their native folds under extreme conditions is still not fully understood. Recent computational and structural studies have identified disulfide bonding as an important mechanism for stabilizing intracellular proteins in certain thermophilic microbes. Here, we present the first proteomic analysis of intracellular disulfide bonding in the hyperthermophilic archaeon Pyrobaculum aerophilum. Our study reveals that the utilization of disulfide bonds extends beyond individual proteins to include many protein-protein complexes. We report the 1.6 A crystal structure of one such complex, a citrate synthase homodimer. The structure contains two intramolecular disulfide bonds, one per subunit, which result in the cyclization of each protein chain in such a way that the two chains are topologically interlinked, rendering them inseparable. This unusual feature emphasizes the variety and sophistication of the molecular mechanisms that can be achieved by evolution. 相似文献
999.
The P2X7 receptor (P2X7R) is an ATP-gated ion channel highly expressed in microglia. P2X7R plays important roles in inflammatory responses in the brain. However, little is known about the mechanisms regulating its functions in microglia. Lysophosphatidylcholine (LPC), an inflammatory phospholipid that promotes microglial activation, may have some relevance to P2X7R signaling in terms of microglial function. In this study, we examined its effects on P2X7R signaling in a mouse microglial cell line (MG6) and primary microglia. LPC facilitated the sustained increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) through P2X7R channels activated by ATP or BzATP. The potentiated increase in [Ca(2+)](i) was actually inhibited by P2X7R antagonists, brilliant blue G and oxidized ATP. The potentiating effect of LPC was not observed with P2Y receptor systems, which are also expressed in MG6 cells. G2A, a receptor for LPC, was expressed in MG6 cells, but not involved in the facilitating effect of LPC on the P2X7R-mediated change in [Ca(2+)](i). Furthermore, LPC enhanced the P2X7R-associated formation of membrane pores and the activation of p44/42 mitogen-activated protein kinase. These results suggest that LPC may regulate microglial functions in the brain by enhancing the sensitivity of P2X7R. 相似文献
1000.
We have investigated the mechanisms by which activation of cannabinoid receptors reduces glutamate release from cerebrocortical nerve terminals. Glutamate release evoked by depolarization of nerve terminals with high KCl (30 mmol/L) involves N and P/Q type Ca(2+)channel activation. However, this release of glutamate is independent of Na(+) or K(+) channel activation as it was unaffected by blockers of these channels (tetrodotoxin -TTX- or tetraethylammonium TEA). Under these conditions in which only Ca(2+) channels contribute to pre-synaptic activity, the activation of cannabinoid receptors with WIN55,212-2 moderately reduced glutamate release (26.4 +/- 1.2%) by a mechanism that in this in vitro model is resistant to TTX and consistent with the inhibition of Ca(2+) channels. However, when nerve terminals are stimulated with low KCl concentrations (5-10 mmol/L) glutamate release is affected by both Ca(2+) antagonists and also by TTX and TEA, indicating the participation of Na(+) and K(+) channel firing in addition to Ca(2+) channel activation. Interestingly, stimulation of nerve terminals with low KCl concentrations uncovered a mechanism that further inhibited glutamate release (81.78 +/- 4.9%) and that was fully reversed by TEA. This additional mechanism is TTX-sensitive and consistent with the activation of K(+) channels. Furthermore, Ca(2+) imaging of single boutons demonstrated that the two pre-synaptic mechanisms by which cannabinoid receptors reduce glutamate release operate in distinct populations of nerve terminals. 相似文献